SUMMARY: MicroRNAs are small (19 to 22-nt long) RNAs that are major regulators of gene expression. MicroRNAs bind to specific mRNAs to promote their degradation. A major goal is to discover microRNAs that contribute to cellular and disease processes. The ability to discover microRNAs involved in cellular pathways using unbiased genomic screening is highly limited. In contrast to mRNAs, microRNAs cannot be easily screened in a genome-wide manner for their role in diverse processes using bar-coded lentiviral CRISPR and shRNA libraries. Here we propose a fundamentally new approach that will lead to a highly efficient library for screening all microRNAs encoded by the genome. Our approach is to express a library of circular RNAs that act as ?microRNA sponges,? each tailored to sequester a specific microRNA in the genome. Although circular RNAs are known to be effective microRNA sponges, the inability to express circular RNAs at high levels in cells has limited their ability to sponge microRNAs, which are highly abundant. Chimerna scientists, while working at Cornell University, developed a fundamentally new strategy to express circular RNA at 100X higher levels than any other previous expression system. Because of this powerful and recently developed circular RNA expression system, we can now generate libraries of circular RNA sponges that can systematically sponge each microRNA in the cell. Chimerna scientists have generated key poof-of-concept data demonstrating the efficacy of this approach with a circular RNA that sponges microRNA miR-19. In order to develop and optimize microRNA sponges to make them suitable for creating a genome-wide lentiviral library, the specific aims of this proposal are (1) To optimize circular RNA sponges to achieve maximal depletion of microRNA activity in cells. In this subaim, we will optimize the sponging activity of circular RNA sponges by testing the optimal spacing and the number of microRNA-binding sites. Additionally, microRNA sponges will be developed that can sponge multiple co-regulated microRNAs. Overall, these experiments will lead to optimized RNA circles that are highly effective in sponging one or more microRNA seed families. (2) To systematically test and reduce off-target effects of circular microRNA sponges. In this aim we will optimize circular RNA sponges by reducing any off-target effects. We will use a gene expression analysis approach to quantify the degree of on-target and off-target effects induced by the circular RNA sponges. To remove any off-target effects, we will systematically mutate sequences that may contribute to these effects. Together, these experiments will result in circular RNAs that have reduced off target effects and will therefore be more suitable for developing a microRNA-sponging library. Overall, this project will result in optimized circular RNA sponges with maximal sponging activity and reduced off-target effects on gene expression. The resulting circles, which will be expressed in a lentiviral system, will be suitable for the next phase of this project, which will involve construction of a genome-wide library, targeting over 2000 microRNAs.